Burkholderia pseudomallei causes melioidosis infections and is currently the third leading cause of death in Northeast Thailand. Melioidosis is particularly difficult to treat due to intrinsic resistance to antibiotics. Despite the prevalence of melioidosis there is currently a limited understanding of B. pseudomallei pathogenesis. This research program is focused on a virulence regulator in B. pseudomallei, BpsR4. BpsR4 is a member of the LuxR family of transcriptional regulators that are involved in a type of bacterial communication called quorum sensing. Typically LuxR proteins induce target gene expression in response to acyl-homoserine lactone (AHL) quorum-sensing signals. However, BpsR4 induces transcription of virulence genes in a manner that is AHL-independent. Interestingly, BpsR4 is activated by antibiotics (trimeothoprim and piperacillin) that regulate BpsR4 at the transcriptional level. To our knowledge BpsR4 is the first conserved LuxR-family protein that is AHL-independent, and the role of antibiotics in activating BpsR4 or other LuxR-family proteins is totally unknown. Although BpsR4 is important for virulence in a C. elegans model host, it is also unknown if BpsR4 induces virulence gene transcription during host infections. Our long-term goal is to understand how LuxR-family proteins promote bacterial survival in different environments, including the host. The objective of this application is to determine how antibiotics induce BpsR4 transcription and the importance of BpsR4 in regulating virulence gene expression in the host. Our central hypothesis is that antibiotics activate BpsR4 through unknown antibiotic-responsive transcriptional regulator(s) and that BpsR4 induces virulence gene expression during C. elegans infection. This proposal aims to i) identify antibiotic-responsive BpsR4 regulators and ii) evaluate BpsR4 induction of virulence genes during host infections. Because BpsR4 is a new class of LuxR-family proteins the studies proposed are expected to expand the current view of the LuxR family. The proposed experiments will also provide us with experimental data critical for building a picture of how antibiotics regulate BpsR4 expression and how BpsR4 promotes virulence during infection. This is significant because the results are expected to increase the currently limited understanding of how B. pseudomallei causes disease, and may ultimately lead to new strategies to control and treat melioidosis.